The present invention concerns the treatment and diagnosis of Staphylococcal infections, particularly those of Staphylococcus aureus, and provides a protein, epitopes of same, and antibodies and other binding and neutralising agents specific against same.
Multiple drug resistance (MDR) is an increasing problem amongst gram-positive bacteria (Banergee, S. N. et al. 1991, Am. J. Med. 91: 865-895; Shaberg, D. R. et al., 1991, Am. J. Med. suppl., 88: 72-75; Gaynes, R. P. et al., 1994, Infect. Dis. Clin. Pract., 6: 452-455), particularly in hospitals. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CNS), particularly methicillin-resistant CNS, prove problematic, being resistant to all penicillins and cephalosporins. Resistance to other agents such as quinolones is widespread (Malabarta, A. et al., 1997, Eur. J. Med. Chem., 32: 459-478; Lewis, K., 1994, TIBS, 19: 119-123; Traub, W. H. et al., 1996, Chemotherapy, 42: 118-132). Treatment is typically effected using vancomycin or teicoplanin. However, resistance to these agents is spreading and so new therapies are needed.
WO 98/01154 discloses the use of bacterial and fungal ABC transporter proteins and neutralising agents specific against same in methods of treatment and diagnosis of the human or animal body. Enterococcal ABC transporter proteins having apparent molecular weights of 97 and 54 kDa are identified as being therapeutically useful, and various epitopes are also identified. Staphylococcal homologues of the IstA and IstB proteins of Bacillus thuringiensis (Menou et al., 1990, J. of Bacteriology, 173: 6689-6696) are also identified, the homologues having apparent molecular weights of 69 and 37 KDA and being immunodominant conserved antigens. Also identified are epitopes of same.
A Staphylococcal ABC transporter protein having an apparent molecular weight of 67 KDA has now been successfully isolated and purified by the present inventor from an epidemic MRSA strain, and has the coding sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. These sequences are partially identified by the S. aureus NCTC 8325 genome sequencing project as contig 1184, contig 1177 and contig 1158 containing amino-terminal sequence data. This protein has not previously been suggested to be an ABC transporter protein, and no diagnostic or therapeutic uses have previously been suggested for it. The protein has a calculated true molecular weight of 60.1 kDa, although post-translational modifications result in its being identified in experiments as having an apparent molecular weight of 67 kDa.
The role of the protein is neither suggested nor disclosed by the IstA and IstB homologues of WO 98/01154 since they have different sequences and different molecular weights. Additionally, the samples from which the IstA and IstB homologues were isolated were peritoneal dialysates rather than the blood and wound cultures used for the present invention (below), and such a purification method could not have led to the present invention since the previously used dialysis step caused a change in the relative proportions of antibody in the dialysate when compared to serum. Similarly, other known prior art does not suggest the role of the protein, nor does it suggest it to have a diagnostic or therapeutic use.
Thus according to the present invention there is provided a Staphylococcal ABC transporter protein having the sequence of SEQ ID NO: 2 or a partially modified form thereof or an immunogenic fragment thereof for use in a method of treatment or diagnosis of the human or animal body.
Immunogenic fragments of the protein include any fragment of the protein which elicit an immune response, and include epitopes (i.e. peptides carrying epitopes). Similarly, analogues (mimotopes) of epitopes may be readily created, the mimotopes having different sequences but displaying the same epitope and thus the term xe2x80x9cimmunogenic fragmentsxe2x80x9d also encompasses immunogenic analogues of the fragments e.g. mimotopes. Epitopes may be readily determined and mimotopes readily designed (Geysen, H. M. et al., 1987, Journal of Immunological Methods, 102: 259-274; Geysen, H. M. et al.,1988, J. Mol. Recognit., 1(1):32-41; Jung, G. and Beck-Sickinger, A. G., 1992, Angew. Chem. Int. Ed. Eng., 31: 367-486).
The scope of the present invention does not extend to other non-Staphylococcal ABC transporter proteins, such as those of WO 98/01154. However, the invention does extend to encompass forms of the protein which have been insubstantially modified (i.e. which have been partially modified), particularly forms of the protein which display the same immunogenic properties as the protein itself.
By xe2x80x9cpartial modificationxe2x80x9d and xe2x80x9cpartially modifiedxe2x80x9d is meant, with reference to amino acid sequences, a partially modified form of the molecule which retains substantially the properties of the molecule from which it is derived, although it may of course have additional functionality. Partial modification may, for example, be by way of addition, deletion or substitution of amino acid residues. Substitutions may be conserved substitutions. Hence the partially modified molecule may be a homologue of the molecules from which it was derived. It may, for example, have at least 70% homology with the molecule from which it was derived. It may for example have at least 80, 90 or 95% homology with the molecule from which it was derived. An example of a homologue is an allelic mutant. Similarly nucleotide sequences encoding the molecule or amino acid sequences may be partially modified to code for any such modifications to an amino acid sequence or molecule. Nucleotide sequences may also of course be modified such that they still code for the same amino acid residues but have a different nucleotide sequence.
The Staphylococcus may be S. aureus or it may for example be a coagulase-negative Staphylococcus, S. epidermidis, S. haemolyticus, S. hyicus or S. saprophyticus. 
An immunogenic fragment may comprise an ATP binding site or a part thereof. Peptides carrying (i.e. displaying) a number of epitopes of the ABC transporter protein have also been identified (below) and thus an immunogenic fragment of the protein may comprise the sequence of SEQ ID NO: 3,4,5,9,10,11 or 12. The epitopes of SEQ ID NOs: 3,4 and 5 are displayed by peptides having the sequences of SEQ ID NOs: 6,7 and 8 respectively, and thus an immunogenic fragment may comprise the sequence of SEQ ID NO: 6,7 or 8. In particular, experiments have shown that peptides having SEQ ID NOs: 6 and 7 which display epitopes having SEQ ID NOs: 3 and 4 are of particular therapeutic use. Peptides having the sequences of SEQ ID NOs: 13 and 14 have also been found to carry epitopes, antibody against which is therapeutic in an animal model (see experiments below) and thus an immunogenic fragment may have the formula of SEQ ID NO: 13 or 14. An additional epitope having the sequence of SEQ ID NO: 17 has also been found, and a peptide having the sequence of SEQ ID NO: 18 carrying same elicits the generation of polyclonal antisera specific against the 67 kDa antigen. Thus an immunogenic fragment may have the sequence of either one of SEQ ID NOs: 17 or 18.
The Staphylococcal ABC transporter protein, displaying epitopes including those described above, therefore provides a therapeutic and diagnostic opportunityxe2x80x94the protein and immunogenic fragments thereof may be used in therapy, both prophylactically (e.g. as immunostimulants such as vaccines) and for treatment of a Staphylococcal infection.
Binding agents and neutralising agents (such as antibodies) specific against the ABC transporter protein, immunogenic fragments thereof or partially modified forms thereof may also be used both diagnostically and therapeutically. Binding agents have a target to which they are specific, and in the case of a binding agent being an antibody, the target is an antigen. An example of a therapeutic medicament is antibody specific against the ABC transporter protein, and this may be employed in immunotherapy, for example passive immunotherapy. Antibodies, their manufacture and use are well known (Harlow, E. and Lane, D., xe2x80x9cAntibodiesxe2x80x94A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1988; Harlow, E. and Lane, D., xe2x80x9cUsing Antibodies: A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory Press, New York, 1998) and so antibodies and antigen binding fragments thereof will be readily apparent to one skilled in the art.
The nucleotide sequence of the protein or immunogenic fragment may also provide the basis for therapeutic applications. For example a nucleotide sequence encoding the protein or immunogenic fragment thereof may be used in the manufacture of a DNA vaccine (Montgomery, D. L. et al., 1997, Pharmacol. Ther., 74(2): 195-205; Donnelly, J. J. et al., 1997, Annu. Rev. Immunol., 15: 617-648; Manickan, E. et al., 1997, Crit. Rev. Immunol., 17(2): 139-154). Other neutralising agents such as ribozymes and antisense oligonucleotides will be readily apparent to one skilled in the art.
Thus the present invention also provides the use of the Staphylococcal ABC transporter protein, immunogenic fragment of same, binding agents and neutralising agents specific against same in a method of manufacture of a medicament for treating Staphylococcal infections. Also provided is a method of manufacture of a medicament for treating Staphylococcal infections, characterised in the use of same. Also provided is a method of treatment of the human or animal body comprising the use of same. The dosage of a medicament may be readily determined by standard dose-response experiments. Medicaments may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient (Remington""s Pharmaceutical Sciences and US Pharmacopeia, 1984, Mack Publishing Company, Easton, Pa., USA).
As discussed above, the ABC transporter protein, immunogenic fragments of same, binding agents and neutralising agents specific against same may also be used diagnostically, and so the present invention provides for their use in the manufacture of a diagnostic test kit for Staphylococci, particularly for Staphylococcal infections. Also provided is their use in a diagnostic test method for Staphylococci.
Also provided according to the present invention is a diagnostic test method for Staphylococcal infection, comprising the steps of:
i) reacting an ABC transporter protein or immunogenic fragment thereof according to the present invention with a sample;
ii) detecting an antibody-antigen binding reaction; and
iii) correlating detection of the antibody-antigen binding reaction with the presence of Staphylococci.
Also provided according to the present invention is a diagnostic test method for Staphylococcal infection, comprising the steps of:
i) reacting an antibody or other binding agent specific against an ABC transporter protein according to the present invention with a sample;
ii) detecting a binding agent-target binding reaction; and
iii) correlating detection of the binding agent-target binding reaction with the presence of Staphylococci.
Samples may be of patient plasma or a fraction thereof e.g. sera or antisera. The diagnostic test method may be for Staphylococcal infection of a patient, the sample being a patient sample and the correlation determining Staphylococcal infection of the patient.
Also provided according to the present invention is a diagnostic test kit for performing a diagnostic test method according to the present invention. The diagnostic test kit may include instructions for performing a diagnostic test using the kit.
Also provided according to the present invention is a method of treatment or diagnosis of Staphylococcal infection comprising the use of a Staphylococcal ABC transporter protein, immunogenic fragment thereof, binding agent or neutralising agent according to the present invention.